Periodic Reporting for period 1 - KNOVV (Kidney Nephron Orientation in Vivo-like in Vitro)
Berichtszeitraum: 2023-04-01 bis 2025-09-30
Current knowledge allows researchers to generate renal organoids - cellular aggregates that resemble in vivo organs. They contain many specific cell types and structures, but they are not correctly organized in space. This may limit their research utility and clinical applications. Therefore, the aim of KNOVV project was to understand if it is possible to micro-manipulate kidney development with small molecules in order to achieve highly specific spatial orientation of renal structures.
The first aim of this project was to understand if we can generate renal progenitors from human induced pluripotent stem cells (hiPSC), that would be able to develop into functional kidneys. We used established protocols and modified them to suit our hiPSC cell line to obtain good quality of metanephric mesenchyme (MM, renal progenitor giving rise to nephrons) and ureteric bud (UB, renal progenitors giving rise to ureter) cells and combined them together to form organoids (MM+UB). These cells presented cross-talk similar to the one already observed and described in mouse kidney rudiments cultures, which resulted in very good development. The correct renal structures formation was confirmed on a gene level (qPCR) and in whole-mount immunofluorescent staining. Those organoids, did show good overall development where nephron – basic renal unit – segments developed in a specific order: glomeruli were connected with proximal tubules, those to distal tubules, and those to collecting ducts. Moreover, when we tested the functionality of proximal tubules, they did show uptake of small fluorescently labelled molecules directed by organic anion transporters located in the proximal tubule membranes.
Next, in order to modify the spatial nephron development, we selected five molecules that would affect the NOTCH pathway. This pathway is known to regulate development of proximal end of the nephron, including glomerulus and proximal tubules. Three molecules were described as inhibiting NOTCH pathway and therefore should inhibit glomerulogenesis and proximal tubule development, while two molecules were described as enhancing NOTCH pathway, which should increase glomerulogenesis and proximal tubule development. Neither of them was previously tested on renal cells or samples/organoids. We tested these molecules using advanced HPLC methodology and various concentrations of those molecules effects on MM spheroids only, UB spheroids only and MM+UB organoids were characterized using gene expression methods (qPCR and Western Blot) and immunofluorescent stainings. Those analyses revealed that only one molecule out of five initially selected, performed to expected standards. This included lack of negative impact on either cell type (MM or UB) and rapid effect – with only 24h treatment we observed expected effect.
Another aim of the KNOVV project was to test novel Start-PEG hydrogel ability to bioprint and provide structural support for renal organoids without having negative impact on their development. Multiple analyses showed that it was: (i) possible to bioprint the hydrogel into fine, easy to manipulate fibers; (ii) hydrogel did provide the structural support for the bioprinted renal organoids; and (iii) those organoids developed very well presenting renal structures similar to control (non-bioprinted) samples. Moreover, it was possible to functionalize the hydrogel with the chosen molecules to affect NOTCH pathway. Those molecules were released from the hydrogel within 30h and remained active (non-degraded).
Next, in order to modify the spatial nephron development, we selected five molecules that would affect the NOTCH pathway. This pathway is known to regulate development of proximal end of the nephron, including glomerulus and proximal tubules. Three molecules were described as inhibiting NOTCH pathway and therefore should inhibit glomerulogenesis and proximal tubule development, while two molecules were described as enhancing NOTCH pathway, which should increase glomerulogenesis and proximal tubule development. Neither of them was previously tested on renal cells or samples/organoids. We tested these molecules using advanced HPLC methodology and various concentrations of those molecules effects on MM spheroids only, UB spheroids only and MM+UB organoids were characterized using gene expression methods (qPCR and Western Blot) and immunofluorescent stainings. Those analyses revealed that only one molecule out of five initially selected, performed to expected standards. This included lack of negative impact on either cell type (MM or UB) and rapid effect – with only 24h treatment we observed expected effect.
Another aim of the KNOVV project was to test novel Start-PEG hydrogel ability to bioprint and provide structural support for renal organoids without having negative impact on their development. Multiple analyses showed that it was: (i) possible to bioprint the hydrogel into fine, easy to manipulate fibers; (ii) hydrogel did provide the structural support for the bioprinted renal organoids; and (iii) those organoids developed very well presenting renal structures similar to control (non-bioprinted) samples. Moreover, it was possible to functionalize the hydrogel with the chosen molecules to affect NOTCH pathway. Those molecules were released from the hydrogel within 30h and remained active (non-degraded).
Despite the progress achieved in this project, it was not possible to complete the bioprinting the renal organoids in the functionalized hydrogel and showing improved spatial nephron orientation, due to time constraints. We made an incredible progress in developmental biology and bioprinting field.
We established that:
1. We can generate functional renal organoids by mixing early progenitors
2. Star-PEG hydrogel:
a. supports the renal organoid development
b. is printable using microfluidic bioprinter
c. provides structural support for bioprinted renal organoids
d. can be functionalized with small molecules
3. we identified new molecules that modified renal organoids development
a. by affecting only developing nephrons
b. not affecting ureteric bud (collecting duct) development
c. being released from the hydrogel during different periods.
The collaborating company, TissueGUARD, is already commercializing the hydrogel, however, more research is needed to further validate the functionalized with molecules hydrogel in order to pursue patent application.
Those findings, will enable future researchers to advance these fields further.
We established that:
1. We can generate functional renal organoids by mixing early progenitors
2. Star-PEG hydrogel:
a. supports the renal organoid development
b. is printable using microfluidic bioprinter
c. provides structural support for bioprinted renal organoids
d. can be functionalized with small molecules
3. we identified new molecules that modified renal organoids development
a. by affecting only developing nephrons
b. not affecting ureteric bud (collecting duct) development
c. being released from the hydrogel during different periods.
The collaborating company, TissueGUARD, is already commercializing the hydrogel, however, more research is needed to further validate the functionalized with molecules hydrogel in order to pursue patent application.
Those findings, will enable future researchers to advance these fields further.